Reversible protein phosphorylation is one of the most pervasive strategies for regulation in biological systems. The phosphorylation of isocitrate dehydrogenase (IDH) of Escherichia coli is an example of this form of regulation. The ultimate goal of this project is to understand the functioning of this phosphorylation system in the intact organism. This proposal focuses on the structure/function relationships of IDH kinase and IDH phosphatase and the regulation of these activities in vivo. IDH kinase and phosphatase have been found to be physically associated, probably on the same polypeptide chain. In an effort to determine the regulatory and structural significance of this bifunctional structure, the relationships between the functional domains will be characterized. In addition to the methods of protein chemistry, this study will make extensive use of recombinant DNA technology, including in vitro mutagenesis. Specific problems to be addressed include the evolutionary relationship between the domains, the degree to which they are physically autonomous and the potential for inter-domain allosteric communication. During the analysis of the structure of IDH kinase/phosphatase, a variety of normal and mutant clones of the gene encoding this protein are being constructed. These clones provide an ideal approach to the study of the IDH phosphorylation cycle in vivo. The cellular level and properties of IDH kinase/phosphatase will be manipulated using these cones and the effects of these perturbations will be characterized. The results of these experiments will provide the basis for the construction of models which describe the regulation of IDH phosphorylation in the intact organism.